Interaction of fascin and protein kinase C : a novel intersection in cell adhesion and motility

Abstract

N.Anilkumar and M.Parsons contributed equally to this workCoordination of protrusive and contractile cell±matrix contacts is important for cell adhesion and migration, but the mechanisms involved are not well understood. We report an unexpected direct association between fascin, an actin-bundling component of ®lopodia, microspikes and lamellipodial ribs, and protein kinase Ca (PKCa), a regulator of focal adhesions. The association is detectable by protein±protein binding in vitro, by coimmunoprecipitation from cell extracts, and in live cells as¯uorescence resonance energy transfer detected by¯uorescence imaging lifetime microscopy. The interaction is physiologically regulated by the extracellular matrix context of cells, depends on activation of PKCa and is mediated by the C1B domain of PKCa. Strikingly, a fascin mutant, fascin S39D, associates constitutively with PKCa. Through use of a newly developed set of membranepermeable peptides that separately inhibit either fascin/PKCa or fascin/actin binding, we have uncovered that speci®c blockade of the fascin/PKCa interaction increases cell migration on ®bronectin in conjunction with increased fascin protrusions and remodeling of focal adhesions. These results identify the fascin±PKCa interaction as an important novel intersection in the regulation and networking of cell± matrix contacts.

Targeted inhibition of fascin function blocks tumour invasion and metastatic colonization

“…Therefore, these data immediately reveal a potential mechanism of action for G2: this inhibitor occupies the actin-binding site 1, prevents the binding of actin filaments to this actin-binding site, and thus leads to the inhibition of the bundling activity of fascin. In addition to directly binding to actin filaments, fascin also directly interacts with other proteins including LIMK [47][48][49] . To investigate whether G2 could also interfere the interaction of fascin with these proteins, we performed the glutathione S-transferase (GST)-pull-down experiments with GST-fascin fusion protein and whole-cell lysates in the absence and presence of G2 ( Fig.…”

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

“…In particular, we focus on those phosphorylation sites on PKCs that are inducible or regulated in cells (Figure 3). PKC : PKC undergoes phosphorylation at Thr-250 (numbering of amino acids from human sequences are used throughout this manuscript) located in the C2 domain in response to phorbol esters that mimics DAG signalling [95] and also in response to cells adhering to fibronectin [107] (Figure 3). Thr-250 is an autophosphorylation site and hence the phosphorylation status of this isoform at this site has been used as a marker of PKC catalytic activity [95].…”

Dual Actin-bundling and Protein Kinase C-binding Activities of Fascin Regulate Carcinoma Cell Migration Downstream of Rac and Contribute to Metastasis

“…Indirect immunofluorescent stainings for fascin, ␣-actinin, phosphotyrosine, and vinculin were carried out as described previously (Adams, 1997;Anilkumar et al, 2003). Samples labeled with FITC, tetramethylrhodamine B isothiocyanate, or Alexa 568 fluorochromes were examined at room temperature under a Leica DM RXE(TCS-SP/SP-AOSB) confocal laser scanning microscope with HCX Plan Apo 63ϫ numerical aperture 1.4 oil immersion objective lens, using Leica confocal software version 2.5 (Leica, Wetzler, Germany).…”

“…In vitro, fascin cross-links filamentous actin (Factin) into tightly packed, parallel bundles in cooperation with Arp2/3 complex and Wiskott-Aldrich syndrome protein Haviv et al, 2006). In intact cells, fascin-and-actin bundles support cortical cell protrusions and growth cone filopodia Adams et al, 1999;Cohan et al, 2001, Adams andAnilkumar et al, 2003;Svitkina et al, 2003;Vignjevic et al, 2006). Fascin contains N-and C-terminal actin-binding sites and the actin cross-linking activity of fascin is negatively regulated by a protein kinase C (PKC) phosphorylation site (serine-39 in human fascin) that lies within the amino-terminal actin-binding site (Ono et al, 1997).…”